Agonist-dependent action of the juvenile hormone receptor.

Juvenile hormone (JH) signaling is effected at the gene regulatory level by receptors of the bHLH-PAS transcription factor family. The sesquiterpenoid hormones and their synthetic mimics are agonist ligands of a unique JH receptor (JHR) protein, methoprene-tolerant (MET). Upon binding an agonist to its PAS-B cavity, MET dissociates from a cytoplasmic chaperone complex including HSP83 and concomitantly switches to a bHLH-PAS partner taiman, forming a nuclear, transcriptionally active JHR heterodimer. This course of events resembles the vertebrate aryl hydrocarbon receptor (AHR), activated by a plethora of endogenous and synthetic compounds. Like in AHR, the pliable PAS-B cavity of MET adjusts to diverse ligands and binds them through similar mechanisms. Despite recent progress, we only begin to discern agonist-induced conformational shifts within the PAS-B domain, with the ultimate goal to understand how these localized changes stimulate assembly of the active JHR complex, and thus fully grasp the mechanism of JHR signaling.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Ligand-dependent protein interactions of the juvenile hormone receptor captured in real time.

Juvenile hormone (JH) signalling provides vital regulatory functions during insect development via transcriptional regulation of genes critical for the progression of metamorphosis and oogenesis. Despite the importance of JH signalling, the underlying molecular mechanisms remain largely unknown. Our current understanding of the pathway depends on static end-point information and suffers from the lack of time-resolved data. Here, we have addressed the dynamic aspect of JH signalling by monitoring in real time the interactions of insect JH receptor proteins. Use of two tags that reconstitute a functional luciferase when in proximity enabled us to follow the rapid assembly of a JH receptor heterodimer from basic helix-loop-helix/Per-Arnt-SIM (bHLH-PAS) proteins, methoprene-tolerant (Met) and taiman (Tai), upon specific JH binding to Met. On a similar timescale (minutes), the dissociation of Met-Met complexes occurred, again strictly dependent on Met interaction with specific agonist ligands. To resolve questions regarding the regulatory role of the chaperone Hsp90/83 in the JHR complex formation, we used the same technique to demonstrate that the Met-Hsp83 complex persisted in the agonist absence but readily dissociated upon specific binding of JH to Met. Preincubation with the Hsp90 inhibitor geldanamycin showed that the chaperone interaction protected Met from degradation and was critical for Met to produce the active signalling dimer with Tai. Thus, the JH receptor functions appear to be governed by principles similar to those regulating the aryl hydrocarbon receptor, the closest vertebrate homologue of the arthropod JH receptor.

Unique peptidic agonists of a juvenile hormone receptor with species-specific effects on insect development and reproduction.

Juvenile hormones (JHs) control insect metamorphosis and reproduction. JHs act through a receptor complex consisting of methoprene-tolerant (Met) and taiman (Tai) proteins to induce transcription of specific genes. Among chemically diverse synthetic JH mimics (juvenoids), some of which serve as insecticides, unique peptidic juvenoids stand out as being highly potent yet exquisitely selective to a specific family of true bugs. Their mode of action is unknown. Here we demonstrate that, like established JH receptor agonists, peptidic juvenoids act upon the JHR Met to halt metamorphosis in larvae of the linden bug, Pyrrhocoris apterus. Peptidic juvenoids induced ligand-dependent dimerization between Met and Tai proteins from P. apterus but, consistent with their selectivity, not from other insects. A cell-based split-luciferase system revealed that the Met-Tai complex assembled within minutes of agonist presence. To explore the potential of juvenoid peptides, we synthesized 120 new derivatives and tested them in Met-Tai interaction assays. While many substituents led to loss of activity, improved derivatives active at sub-nanomolar range outperformed hitherto existing peptidic and classical juvenoids including fenoxycarb. Their potency in inducing Met-Tai interaction corresponded with the capacity to block metamorphosis in P. apterus larvae and to stimulate oogenesis in reproductively arrested adult females. Molecular modeling demonstrated that the high potency correlates with high affinity. This is a result of malleability of the ligand-binding pocket of P. apterus Met that allows larger peptidic ligands to maximize their contact surface. Our data establish peptidic juvenoids as highly potent and species-selective novel JHR agonists.

Binding of de novo synthesized radiolabeled juvenile hormone (JH III) by JH receptors from the Cuban subterranean termite Prorhinotermes simplex and the German cockroach Blattella germanica.

Juvenile hormone (JH) controls insect reproduction and development through an intracellular receptor complex comprising two bHLH-PAS proteins, the JH-binding Methoprene-tolerant (Met) and its partner Taiman (Tai). Many hemimetabolous insects including cockroaches strictly depend on JH for stimulation of vitellogenesis. In termites, the eusocial hemimetabolans, JH also regulates the development of caste polyphenism. Studies addressing the agonist ligand binding to recombinant JH receptors currently include three species belonging to two holometabolous insect orders, but none that would represent any of the hemimetabolous orders. Here, we examined JH receptors in two representatives of Blattodea, the cockroach Blattella germanica and the termite Prorhinotermes simplex. To test the JH-binding capacity of Met proteins from these species, we performed chemical synthesis and tritium labeling of the natural blattodean JH homolog, JH III. Our improved protocol increased the yield and specific activity of [10-3H]JH III relative to formerly available preparations. Met proteins from both species specifically bound [3H]JH III with high affinity, whereas Met variants mutated at a critical position within the ligand-binding domain were incapable of such binding. Furthermore, JH III and the synthetic JH mimic fenoxycarb stimulated dimerization between Met and Tai components of the respective JH receptors of both species. These data present primary evidence for agonist binding by JH receptors in any hemimetabolous species and provide a molecular basis for JH action in cockroaches and termites.

Effects of quercetin and metabolites on uric acid biosynthesis and consequences for gene expression in the endothelium.

BACKGROUND: Uric acid, a metabolic product of purine degradation in humans, is a risk factor for developing gout and type 2 diabetes, and supplementation with quercetin lowers plasma uric acid in mildly hyperuricemic men. Here we examined the mechanism of inhibition of enzymes involved in uric acid metabolism by quercetin, conjugates and microbial catabolites, and measured the effect of lowered circulating uric acid on endothelial cell gene expression. METHODS: Inhibition of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) and xanthine oxidoreductase (XOR) activity by quercetin and metabolites was determined by HPLC. Human umbilical vein endothelial cells (HUVECs) were cultured under conditions mimicking blood flow, treated with uric acid (0, 300 or 500 µmol/L), and changes in gene expression measured using transcriptomics and quantitative droplet digital PCR. RESULTS: In human plasma, no inhibition of PNP activity was observed, and only quercetin weakly inhibited ADA. XOR was not present at sufficient amount in human plasma to use for testing, but quercetin, quercetin-3'-sulfate and the gut microbial metabolite 3',4'-dihydroxyphenylacetic acid inhibited bovine milk XOR. Several changes were observed in gene expression in HUVECs under flow compared to static conditions, but after uric acid treatment, only very few changes were detected. CONCLUSIONS: We propose that the main mechanism by which quercetin, as quercetin-3'-sulfate, lowers uric acid in vivo is through inhibition of XOR, and not ADA nor PNP. The pertinent shift in uric acid concentration was not sufficient to produce significant changes in endothelial gene expression in a cell model.

Bridgehead Modifications of Englerin A Reduce TRPC4 Activity and Intravenous Toxicity but not Cell Growth Inhibition.

Modifications at the bridgehead position of englerin A were made to explore the effects of variation at this site on the molecule for biological activity, as judged by the NCI 60 screen, in which englerin A is highly potent and selective for renal cancer cells. Replacement of the isopropyl group by other, larger substituents yielded compounds which displayed excellent selectivity and potency comparable to the natural product. Selected compounds were also evaluated for their effect on the ion channel TRPC4 as well as for intravenous toxicity in mice, and these had lower potency in both assays compared to englerin A.

The effect of quercetin on endothelial cells is modified by heterocellular interactions.

Single cell-type models are useful for determining mechanisms, but in vivo, cell-cell interactions are important, and neighbouring cells can impact endothelial cell function. Quercetin can attenuate endothelial dysfunction by modulating vascular tone and reducing inflammation. We determined the effect of quercetin on a co-culture between Human Umbilical Vein Endothelial Cells (HUVEC) and human HepG2 hepatic cells or human LHCN-M2 muscle cells. Heme oxygenase-1 (HO-1) mRNA and protein were decreased, pyruvate dehydrogenase kinase (PDK) 4 and glucose transporter (GLUT) 3 mRNA increased, and GLUT1 protein decreased in HUVEC when cultured with HepG2. GLUT transporters, but not the other targets, were similarly regulated in co-culture with muscle cells. Some but not all of the effects were mediated by lactate and transforming growth factor ß1. Quercetin added apically to the endothelial cells upregulated HO-1 and downregulated PDK4 both in monoculture and in co-culture, but the total PDK4 levels were higher in the presence of HepG2 cells. In the absence of general permeability changes, glucose transport across the endothelial monolayer was elevated in the presence of HepG2 cells, however this effect was moderated by quercetin applied on the apical side of the endothelial cells. At lower glucose concentration, apical quercetin also promoted glucose uptake in HepG2 cells. Co-culturing HUVEC with the HepG2 cells showed capacity to modulate quercetin-elicited changes in endothelial gene transcription and glucose transport.

Indirect Chronic Effects of an Oleuropein-Rich Olive Leaf Extract on Sucrase-Isomaltase In Vitro and In Vivo.

Consumption of dietary bioactives is an avenue to enhancing the effective healthiness of diets by attenuating the glycaemic response. The intestinal brush border enzyme sucrase-isomaltase (SI) is the sole enzyme hydrolysing consumed sucrose, and we previously showed the acute effects of olive leaf extract (OLE) on sucrase activity when given together with sugars both in vitro and in vivo. Here we tested whether OLE could affect sucrase expression when pre-incubated chronically, a "priming" effect not dependent on competitive interaction with SI, in both a cell model and a human intervention. Using differentiated Caco-2/TC7 cells, long-term pre-treatment with oleuropein-rich olive leaf extract (OLE) lowered SI mRNA, surface protein and activity, and attenuated subsequent sucrose hydrolysis. Based on these results, a randomised, double-blinded, placebo-controlled, crossover pilot study was conducted. OLE (50 mg oleuropein) was consumed in capsule form 3 times a day for 1 week by 11 healthy young women followed by an oral sucrose tolerance test in the absence of OLE. However this treatment, compared to placebo, did not induce a change in post-prandial blood glucose maximum concentration (Glcmax), time to reach Glcmax and incremental area under the curve. These results indicate that changes in SI mRNA, protein and activity in an intestinal cell model by OLE are not sufficient under these conditions to induce a functional effect in vivo in healthy volunteers.

Inhibition of intestinal glucose transport by polyphenols: a mechanism for indirect attenuation of cholesterol absorption?

Cholesterol uptake and chylomicron synthesis are promoted by increasing glucose concentrations in both healthy and diabetic individuals during the postprandial phase. The goal of this study was to test whether acute inhibition of glucose uptake could impact cholesterol absorption in differentiated human intestinal Caco-2 cells. As expected, high glucose upregulated intestinal cholesterol metabolism promoting its uptake and incorporation in lipoproteins. This was accompanied by an increase in the gene expression of Niemann-Pick C1 Like 1 and proprotein convertase subtillisin/kexin type 9. Cholesterol uptake was attenuated by acute inhibition of glucose absorption by cytochalasin B, by a chamomile extract and by one of its main constituent polyphenols, apigenin 7-O-glucoside; however, chylomicron secretion was only reduced by the chamomile extract. These data support a potential indirect role for bioactives in modulating intestinal lipid pathways through effects on intestinal glucose uptake. This working hypothesis warrants further testing in an in vivo setting such as in hypercholesterolaemic or prediabetic individuals.

Long term treatment with quercetin in contrast to the sulfate and glucuronide conjugates affects HIF1α stability and Nrf2 signaling in endothelial cells and leads to changes in glucose metabolism.

Endothelial functionality profoundly contributes to cardiovascular health. The effects of flavonoids shown to improve endothelial performance include regulating blood pressure by modulating endothelial nitric oxide synthase and NADPH oxidases, but their impact on glucose uptake and metabolism has not been explored. We treated human umbilical vein endothelial cells (HUVEC) with the flavonoid quercetin and its circulating metabolites acutely and chronically, then assessed glucose uptake, glucose metabolism, gene transcription and protein expression. Acute treatment had no effect on glucose uptake, ruling out any direct interaction with sugar transporters. Long term treatment with quercetin, but not quercetin 3-O-glucuronide or 3'-O-sulfate, significantly increased glucose uptake. Heme oxygenase-1 (HO-1) was induced by quercetin but not its conjugates, but was not implicated in the glucose uptake stimulation since hemin, a classical inducer of HO-1, did not affect glucose metabolism. Quercetin increased stability of the transcription factor hypoxia induced factor 1α (HIF1α), a powerful stimulant of glucose metabolism, which was also paralleled by treatment with a prolyl-4-hydroxylase inhibitor dimethyloxalylglycine (DMOG). 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which regulates the rate of glycolysis, was upregulated by both quercetin and DMOG. Pyruvate dehydrogenase kinase (PDK) isoforms regulate pyruvate dehydrogenase; PDK2 and PDK4 were down-regulated by both effectors, but only DMOG also upregulated PDK1 and PDK3. Quercetin, but not DMOG, increased glucose-6-phosphate dehydrogenase. Chronic quercetin treatment also stimulated glucose transport across the HUVEC monolyer in a 3D culture model. Gene expression of several flavonoid transporters was repressed by quercetin, but this was either abolished (Organic anion transporter polypeptide 4C1) or reversed (Multidrug resistance gene 1) by both conjugates. We conclude that quercetin and its circulating metabolites differentially modulate glucose uptake/metabolism in endothelial cells, through effects on HIF1α and transcriptional regulation of energy metabolism.

Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism.

The gut microbiome supplies essential metabolites such as short-chain fatty acids to skeletal muscle mitochondria, and the composition and activity of the microbiota is in turn affected by muscle fitness. To further our understanding of the complex interactions between the gut microbiome and muscle, we examined the effect of microbiota-derived phenolic metabolites on the ability of human muscle cells to take up and metabolize glucose. As a model, we used the differentiated human skeletal muscle myoblast line, LHCN-M2, which expresses typical muscle phenotypic markers. We initially tested a selected panel of parent phenolic compounds and microbial metabolites, and their respective phenolic conjugates, as found in blood. Several of the tested compounds increased glucose uptake and metabolism, notably in high glucose- and insulin-treated myotubes. One of the most effective was isovanillic acid 3 -O-sulfate (IVAS), a metabolite from the microbiome found in the blood, primarily derived from consumed cyanidin 3 -O-glucoside, a major compound in berry fruits. IVAS stimulated a dose-dependent increase in glucose transport through glucose transporter GLUT4- and PI3K-dependent mechanisms. IVAS also up-regulated GLUT1, GLUT4, and PI3K p85α protein, and increased phosphorylation of Akt. The stimulation of glucose uptake and metabolism by a unique microbiome metabolite provides a novel link among diet, gut microbiota, and skeletal muscle energy source utilization.-Houghton, M. J., Kerimi, A., Mouly, V., Tumova, S., Williamson, G. Gut microbiome catabolites as novel modulators of muscle cell glucose metabolism.

Quercetin preserves redox status and stimulates mitochondrial function in metabolically-stressed HepG2 cells.

Hyperglycemia augments formation of intracellular reactive oxygen species (ROS) with associated mitochondrial damage and increased risk of insulin resistance in type 2 diabetes. We examined whether quercetin could reverse chronic high glucose-induced oxidative stress and mitochondrial dysfunction. Following long-term high glucose treatment, complex I activity was significantly decreased in isolated mitochondria from HepG2 cells. Quercetin dose-dependently recovered complex I activity and lowered cellular ROS generation under both high and normal glucose conditions. Respirometry studies showed that quercetin could counteract the detrimental increase in inner mitochondrial membrane proton leakage resulting from high glucose while it increased oxidative respiration, despite a decrease in electron transfer system (ETS) capacity, and lower non-ETS oxygen consumption. A quercetin-stimulated increase in cellular NAD+/NADH was evident within 2 h and a two-fold increase in PGC-1α mRNA within 6 h, in both normal and high glucose conditions. A similar pattern was also found for the mRNA expression of the repulsive guidance molecule b (RGMB) and its long non-coding RNA (lncRNA) RGMB-AS1 with quercetin, indicating a potential change of the glycolytic phenotype and suppression of aberrant cellular growth which is characteristic of the HepG2 cells. Direct effects of quercetin on PGC-1α activity were minimal, as quercetin only weakly enhanced PGC-1α binding to PPARα in vitro at higher concentrations. Our results suggest that quercetin may protect mitochondrial function from high glucose-induced stress by increasing cellular NAD+/NADH and activation of PGC-1α-mediated pathways. Lower ROS in combination with improved complex I activity and ETS coupling efficiency under conditions of amplified oxidative stress could reinforce mitochondrial integrity and improve redox status, beneficial in certain metabolic diseases.

Acute metabolic actions of the major polyphenols in chamomile: an in vitro mechanistic study on their potential to attenuate postprandial hyperglycaemia.

Transient hyperglycaemia is a risk factor for type 2 diabetes and endothelial dysfunction, especially in subjects with impaired glucose tolerance. Nutritional interventions and strategies for controlling postprandial overshoot of blood sugars are considered key in preventing progress to the disease state. We have identified apigenin-7-O-glucoside, apigenin, and (Z) and (E)-2-hydroxy-4-methoxycinnamic acid glucosides as the active (poly)phenols in Chamomile (Matricaria recutita) able to modulate carbohydrate digestion and absorption in vitro as assessed by inhibition of α-amylase and maltase activities. The latter two compounds previously mistakenly identified as ferulic acid hexosides were purified and characterised and studied for their contribution to the overall bioactivity of chamomile. Molecular docking studies revealed that apigenin and cinnamic acids present totally different poses in the active site of human α-amylase. In differentiated Caco-2/TC7 cell monolayers, apigenin-7-O-glucoside and apigenin strongly inhibited D-[U-14C]-glucose and D-[U-14C]-sucrose transport, and less effectively D-[U-14C]-fructose transport. Inhibition of D-[U-14C]-glucose transport by apigenin was stronger under Na+-depleted conditions, suggesting interaction with the GLUT2 transporter. Competitive binding studies with molecular probes indicate apigenin interacts primarily at the exofacial-binding site of GLUT2. Taken together, the individual components of Chamomile are promising agents for regulating carbohydrate digestion and sugar absorption at the site of the gastrointestinal tract.

Differential patterns of inhibition of the sugar transporters GLUT2, GLUT5 and GLUT7 by flavonoids.

Only limited data are available on the inhibition of the sugar transporter GLUT5 by flavonoids or other classes of bioactives. Intestinal GLUT7 is poorly characterised and no information exists concerning its inhibition. We aimed to study the expression of GLUT7 in Caco-2/TC7 intestinal cells, and evaluate inhibition of glucose transport by GLUT2 and GLUT7, and of fructose transport by GLUT2, GLUT5 and GLUT7, by flavonoids. Differentiated Caco-2/TC7 cell monolayers were used to investigate GLUT7 expression, as well as biotinylation and immunofluorescence to assess GLUT7 location. For mechanistic sugar transport studies, X. laevis oocytes were injected with individual mRNA, and GLUT protein expression on oocyte membranes was confirmed. Oocytes were incubated with D-[14C(U)]-glucose or D-[14C(U)]-fructose in the presence of flavonoids, and uptake was estimated by liquid scintilation counting. In differentiated Caco-2/TC7 cell monolayers, GLUT7 was mostly expressed apically. When applied apically, or to both compartments, sorbitol, galactose, L-glucose or sucrose did not affect GLUT7 mRNA expression. Fructose applied to both sides increased GLUT7 mRNA (13%, p ≤ 0.001) and total GLUT7 protein (2.7-fold, p ≤ 0.05), while the ratio between apical, basolateral and total GLUT7 protein was unchanged. In the X. laevis oocyte model, GLUT2-mediated glucose and fructose transport were inhibited by quercetin, (-)-epigallocatechin gallate (EGCG) and apigenin, GLUT5-mediated fructose transport was inhibited by apigenin and EGCG, but not by quercetin, and GLUT7-mediated uptake of both glucose and fructose was inhibited by apigenin, but not by quercetin nor EGCG. Expression of GLUT7 was increased by fructose, but only when applied to Caco-2/TC7 cells both apically and basolaterally. Since GLUT2, GLUT5 and GLUT7 show different patterns of inhibition by the tested flavonoids, we suggest that they have the potential to be used as investigational tools to distinguish sugar transporter activity in different biological settings.

Chronic exposure to short-chain fatty acids modulates transport and metabolism of microbiome-derived phenolics in human intestinal cells.

Dietary fiber-derived short-chain fatty acids (SCFA) and phenolics produced by the gut microbiome have multiple effects on health. We have tested the hypothesis that long-term exposure to physiological concentrations of SCFA can affect the transport and metabolism of (poly)phenols by the intestinal epithelium using the Caco-2 cell model. Metabolites and conjugates of hesperetin (HT) and ferulic acid (FA), gut-derived from dietary hesperidin and chlorogenic acid, respectively, were quantified by LC-MS with authentic standards following transport across differentiated cell monolayers. Changes in metabolite levels were correlated with effects on mRNA and protein expression of key enzymes and transporters. Propionate and butyrate increased both FA transport and rate of appearance of FA glucuronide apically and basolaterally, linked to an induction of MCT1. Propionate was the only SCFA that augmented the rate of formation of basolateral FA sulfate conjugates, possibly via basolateral transporter up-regulation. In addition, propionate enhanced the formation of HT glucuronide conjugates and increased HT sulfate efflux toward the basolateral compartment. Acetate treatment amplified transepithelial transport of FA in the apical to basolateral direction, associated with lower levels of MCT1 protein expression. Metabolism and transport of both HT and FA were curtailed by the organic acid lactate owing to a reduction of UGT1A1 protein levels. Our data indicate a direct interaction between microbiota-derived metabolites of (poly)phenols and SCFA through modulation of transporters and conjugating enzymes and increase our understanding of how dietary fiber, via the microbiome, may affect and enhance uptake of bioactive molecules.

Transendothelial glucose transport is not restricted by extracellular hyperglycaemia.

Endothelial cells are routinely exposed to elevated glucose concentrations post-prandially in healthy individuals and permanently in patients with metabolic syndrome and diabetes, and so we assessed their sugar transport capabilities in response to high glucose. In human umbilical vein (HUVEC), saphenous vein, microdermal vessels and aorta, GLUT1 (SLC2A1), GLUT3 (SLC2A3), GLUT6 (SLC2A6), and in microdermal vessels also GLUT12 (SLC2A12), were the main glucose transporters as assessed by mRNA, with no fructose transporters nor SGLT1 (SLC5A1). Uptake of 14C-fructose was negligible. GLUT1 and GLUT3 proteins were detected in all cell types and were responsible for ~60% glucose uptake in HUVEC, where both GLUT1 and GLUT3, but not GLUT6 siRNA knock-down, reduced the transport. Under shear conditions, GLUT1 protein decreased, GLUT3 increased, and 14C-deoxy-glucose uptake was attenuated. In high glucose, lipid storage was increased, cell numbers were lower, 14C-deoxy-glucose uptake decreased owing to attenuated GLUT3 protein and less surface GLUT1, and trans-endothelial transport of glucose increased due to cell layer permeability changes. We conclude that glucose transport by endothelial cells is relatively resistant to effects of elevated glucose. Cells would continue to supply it to the underlying tissues at a rate proportional to the blood glucose concentration, independent of insulin or fructose.

Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor.

OBJECTIVE: Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca(2+)) entry. Here, we sought understanding of a Synta66-resistant Ca(2+) entry pathway activated by VEGF. APPROACH AND RESULTS: Measurement of intracellular Ca(2+) in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca(2+) entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca(2+) entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca(2+) release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. CONCLUSIONS: VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca(2+) entry and downstream endothelial cell remodeling.

Cellular asymmetric catalysis by UDP-glucuronosyltransferase 1A8 shows functional localization to the basolateral plasma membrane.

UDP-glucuronosyltransferases (UGTs) are highly expressed in liver, intestine and kidney, and catalyze the glucuronic acid conjugation of both endogenous compounds and xenobiotics. Using recombinant human UGT isoforms, we show that glucuronic acid conjugation of the model substrate, (-)-epicatechin, is catalyzed mainly by UGT1A8 and UGT1A9. In HepG2 cells, pretreatment with polyunsaturated fatty acids increased substrate glucuronidation. In the intestinal Caco-2/HT29-MTX co-culture model, overall relative glucuronidation rates were much higher than in HepG2 cells, and (-)-epicatechin was much more readily conjugated when applied to the basolateral side of the cell monolayer. Under these conditions, 95% of the conjugated product was effluxed back to the site of application, and none of the other phase 2-derived metabolites followed this distribution pattern. HT29-MTX cells contained >1000-fold higher levels of UGT1A8 mRNA than Caco-2 or HepG2 cells. Gene expression of UGT1A8 increased after treatment of cells with docosahexaenoic acid, as did UGT1A protein levels. Immunofluorescence staining and Western blotting showed the presence of UGT1A in the basal and lateral parts of the plasma membrane of HT29-MTX cells. These results suggest that some of the UGT1A8 enzyme is not residing in the endoplasmic reticulum but spans the plasma membrane, resulting in increased accessibility to compounds outside the cell. This facilitates more efficient conjugation of substrate and is additionally coupled with rapid efflux by functionally associated basolateral transporters. This novel molecular strategy allows the cell to carry out conjugation without the xenobiotic entering into the interior of the cell.

Expression of a long variant of CRACR2A that belongs to the Rab GTPase protein family in endothelial cells.

CRACR2A protein is described in T cells as an EF-hand-containing modulator of calcium-release-activated calcium (CRAC) channels. Here we sought relevance to calcium entry of endothelial cells. Unexpectedly, short interfering RNA designed to deplete CRACR2A had no effect on CRAC channels in endothelial cells but reduced the abundance of a protein with about twice the mass of CRACR2A. Reference to gene sequence data indicated the potential for a variant transcript encoding a C-terminal Rab GTPase extension of CRACR2A. Full-length cloning demonstrated expression of the long variant in endothelial cells. It was designated CRACR2A-L. Sequence analysis suggested it to be a previously unrecognised member of the Rab GTPase family. It made a positive contribution to endothelial tube formation. The data suggest that endothelial cells contain a long variant of CRACR2A which is an EF-hand-containing Rab protein that lacks impact on CRAC channels.